US8268622B2 - EPSPS mutants - Google Patents

EPSPS mutants Download PDF

Info

Publication number
US8268622B2
US8268622B2 US12/160,725 US16072507A US8268622B2 US 8268622 B2 US8268622 B2 US 8268622B2 US 16072507 A US16072507 A US 16072507A US 8268622 B2 US8268622 B2 US 8268622B2
Authority
US
United States
Prior art keywords
plant
epsps
amino acid
thr
pro
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active, expires
Application number
US12/160,725
Other languages
English (en)
Other versions
US20090307802A1 (en
Inventor
Greg F. W. Gocal
Mark E. Knuth
Peter R. Beetham
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cibus US LLC
Original Assignee
Cibus US LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cibus US LLC filed Critical Cibus US LLC
Priority to US12/160,725 priority Critical patent/US8268622B2/en
Assigned to CIBUS, LLC reassignment CIBUS, LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BEETHAM, PETER R., GOCAL, GREG F.W., KNUTH, MARK E.
Assigned to CIBUS US LLC reassignment CIBUS US LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CIBUS LLC
Publication of US20090307802A1 publication Critical patent/US20090307802A1/en
Assigned to CIBUS US LLC, INCIMA US LLC reassignment CIBUS US LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CIBUS US LLC
Application granted granted Critical
Publication of US8268622B2 publication Critical patent/US8268622B2/en
Assigned to CIBUS US LLC reassignment CIBUS US LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: INCIMA US LLC
Active legal-status Critical Current
Adjusted expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C12N15/8275—Glyphosate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1092—3-Phosphoshikimate 1-carboxyvinyltransferase (2.5.1.19), i.e. 5-enolpyruvylshikimate-3-phosphate synthase

Definitions

  • the present invention relates to the production of a non-transgenic plant resistant or tolerant to an herbicide of the phosphonomethylglycine family, e.g., glyphosate.
  • the present invention also relates to the use of a recombinagenic oligonucleobase to make a desired mutation in the chromosomal or episomal sequences of a plant in the gene encoding for 5-enol pyruvylshikimate-3-phosphate synthase (EPSPS).
  • EPSPS 5-enol pyruvylshikimate-3-phosphate synthase
  • the mutated protein which substantially maintains the catalytic activity of the wild-type protein, allows for increased resistance or tolerance of the plant to a herbicide of the phosphonomethylglycine family, and allows for the substantially normal growth or development of the plant, its organs, tissues or cells as compared to the wild-type plant regardless of the presence or absence of the herbicide.
  • the present invention also relates to an E. coli cell having a mutated EPSPS gene, a non-transgenic plant cell in which the EPSPS gene has been mutated, a non-transgenic plant regenerated therefrom, as well as a plant resulting from a cross using a regenerated non-transgenic plant having a mutated EPSPS gene as one of the parents of the cross.
  • the present mutated EPSPS protein has been changed in amino acid positions 159, 178, 182, 193, 244, 273 and/or 454 in the Arabidopsis EPSPS protein (NM 130093) or at an analogous amino acid residue in an EPSPS paralog.
  • Herbicide-tolerant plants may reduce the need for tillage to control weeds thereby effectively reducing soil erosion.
  • One herbicide which is the subject of much investigation in this regard is N-phosphonomethylglycine, commonly referred to as glyphosate.
  • Glyphosate inhibits the shikimic acid pathway which leads to the biosynthesis of aromatic compounds including amino acids, hormones and vitamins.
  • glyphosate curbs the conversion of phosphoenolpyruvic acid (PEP) and 3-phosphoshikimic acid to 5-enolpyravyl-3-phosphoshikimic acid by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (hereinafter referred to as EPSP synthase or EPSPS).
  • PEP phosphoenolpyruvic acid
  • EPSP synthase enzyme 5-enolpyruvylshikimate-3-phosphate synthase
  • glyphosate includes any herbicidally effective form of N-phosphonomethylglycine (including any salt thereof), other forms which result in the production of the glyphosate anion in plants and any other herbicides of the phosphonomethlyglycine family.
  • Tolerance of plants to glyphosate can be increased by introducing a mutant EPSPS gene having an alteration in the EPSPS amino acid coding sequence into the genome of the plant.
  • Examples of some of the mutations in the EPSPS gene for inducing glyphosate tolerance are described in the following patents: U.S. Pat. No. 5,310,667; U.S. Pat. No. 5,866,775; U.S. Pat. No. 5,312,910; U.S. Pat. No. 5,145,783.
  • the EPSPS gene has a significantly lower enzymatic activity than the wild-type EPSPS.
  • the apparent K m for PEP and the apparent K l for glyphosate for the wild-type EPSPS from E. coli are 10 ⁇ M and 0.5 ⁇ M, respectively, while for a glyphosate-tolerant isolate having a single amino acid substitution of alanine for glycine at position 96, these values are 220 ⁇ M and 4.0 mM, respectively.
  • glyphosate-tolerant EPSPS genes have been constructed by mutagenesis. Again, the glyphosate-tolerant EPSPS had lower catalytic efficiency (V max /K m ), as shown by an increase in the K m for PEP, and a slight reduction of the V max of the wild-type plant enzyme (Kishore et al., 1998, Ann. Rev. Biochem. 57:627-663).
  • Kmiec I Recombinagenic oligonucleobases and their use to effect genetic changes in eukaryotic cells are described in U.S. Pat. No. 5,565,350 to Kmiec (Kmiec I). Kmiec I teaches a method for introducing specific genetic alterations into a target gene. Kmiec I discloses, inter alia, recombinagenic oligonucleobases having two strands, in which a first strand contains two segments of at least 8 RNA-like nucleotides that are separated by a third segment of from 4 to about 50 DNA-like nucleotides, termed an “interposed DNA segment.” The nucleotides of the first strand are base paired to DNA-like nucleotides of a second strand.
  • the first and second strands are additionally linked by a segment of single stranded nucleotides so that the first and second strands are parts of a single oligonucleotide chain.
  • Kmiec I further teaches a method for introducing specific genetic alterations into a target gene. According to Kmiec I, the sequences of the RNA segments are selected to be homologous, i.e., identical, to the sequence of a first and a second fragment of the target gene.
  • the sequence of the interposed DNA segment is homologous with the sequence of the target gene between the first and second fragment except for a region of difference, termed the “heterologous region.”
  • the heterologous region can effect an insertion or deletion, or can contain one or more bases that are mismatched with the sequence of target gene so as to effect a substitution.
  • the sequence of the target gene is altered as directed by the heterologous region, such that the target gene becomes homologous with the sequence of the recombinagenic oligonucleobase.
  • ribose and 2′-O-methylribose, i.e., 2′-methoxyribose, containing nucleotides can be used in recombinagenic oligonucleobases and that naturally-occurring deoxyribose-containing nucleotides can be used as DNA-like nucleotides.
  • U.S. Pat. No. 5,731,181 to Kimec specifically disclose the use of recombinagenic oligonucleobases to effect genetic changes in plant cells and discloses further examples of analogs and derivatives of RNA-like and DNA-like nucleotides that can be used to effect genetic changes in specific target genes.
  • Other patents discussing the use of recombinagenic oligonucleobases include: U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in International Patent No.
  • Recombinagenic oligonucleobases include mixed duplex oligonucleotides, non-nucleotide containing molecules taught in Kmiec II and other molecules taught in the above-noted patents and patent publications.
  • U.S. Pat. No. 6,870,075 discloses a method for producing a non-transgenic, herbicide resistant or tolerant plants employing recombinagenic oligonucleobases according to the methods disclosed in Kmiec I and Kmiec II.
  • the EPSPS mutants disclosed in the '075 patent include changes made in the following amino acid positions of the EPSPS protein: Leu 173 , Gly 177 , Thr 178 , Ala 179 , Met 180 , Arg 181 , Pro 182 , Ser 98 , Ser 255 and Leu 198 in the Arabidopsis EPSPS protein or at an analogous amino acid residue in an EPSPS paralog.
  • the present invention relates to additional amino acid mutations that can be made in any EPSPS gene from any species to produce a gene product that possesses resistance to glyphosate.
  • a non-transgenic plant or plant cell having one or more mutations in the EPSPS gene is made.
  • the resulting plant has increased resistance or tolerance to a member of the phosphonomethylglycine family such as glyphosate and exhibits substantially normal growth or development of the plant, its organs, tissues or cells, as compared to the corresponding wild-type plant or cell.
  • the mutated gene produces a gene product having a substitution at one or more of the amino acid positions 160, 179, 183, 194, 244, 273 and/or 454 in the Arabidopsis EPSPS protein (AF360224) or at an analogous amino acid residue in an EPSPS paralog.
  • the mutated plant is resistant to glyphosate and has substantially the same catalytic activity as compared to the wild-type EPSPS protein.
  • the present invention includes a mutated EPSPS gene from an E. coli and mutated E. coli cells that produces a gene product having a substitution at one or more of the amino acid positions 82, 97, 101, 114, 164, 193 and 374.
  • the mutated E. coli EPSPS gene can be used for in vitro testing of the mutated gene product. Once active E. coli mutants have been identified then corresponding mutants can then be made to an EPSPS gene in a desirable crop to impart herbicide resistance to the crop.
  • the present invention also relates to a method for producing a non-transgenic plant having a mutated EPSPS gene that substantially maintains the catalytic activity of the wild-type protein regardless of the presence or absence of a herbicide of the phosphonomethylglycine family.
  • the method comprises introducing into a plant cell a recombinagenic oligonucleobase with a targeted mutation in the EPSPS gene that produces a gene product having one or more of the aforementioned amino acid changes.
  • the method further includes identifying a cell, seed, or plant having a mutated EPSPS gene and to culturing and regeneration methods to obtain a plant that produces seeds, henceforth a “fertile plant”, and the production of seeds and additional plants from such a fertile plant including descendant (progeny) plants that contain the mutated EPSPS gene.
  • the invention is further directed to a method of selectively controlling weeds in a field.
  • the field comprises plants with the disclosed EPSPS gene alterations and weeds.
  • the method comprises application to the field of a phospnomethyglycine herbicide to which the said plants are rendered resistant and the weeds are controlled.
  • a preferred herbicide is glyphosate.
  • the invention is also directed to novel mutations in the EPSPS gene and resulting novel gene product that confer resistance or tolerance to a member of the phosphonomethylglycine family, e.g., glyphosate, to a plant or wherein the mutated EPSPS has substantially the same enzymatic activity as compared to wild-type EPSPS. Additionally, the present invention is directed to a mutated E. coli EPSPS gene product protein) that is used to screen EPSPS mutants for use as herbicide resistant mutations in plants.
  • FIG. 1 shows the EPSPS gene (AroA gene) product protein sequence (SEQ ID NO: 17) in E. Coli where the mutated amino acid positions are depicted with a box around them. The substituted amino acid in those positions is shown below the sequence.
  • FIG. 2 shows the protein sequence (SEQ ID NO: 18) of AtEPSPS cDNA-At2g45300 translated from Genbank accession NM — 130093 (Arabodopsis).
  • FIG. 3 shows the protein sequence (SEQ ID NO: 19) of AtEPSPS cDNA-At1g48860 translated from Genbank accession AF360224T (Arabodopsis).
  • FIG. 4 shows the protein sequence (SEQ ID NO: 20) of BnEPSPS cDNA-BN-2 2-23 (Canola).
  • FIG. 5 shows the protein sequence (SEQ ID NO: 21) of BNEPSPS cDNA-2-28 from X51475 gDNA translation (Canola).
  • An oligonucleobase is a polymer of nucleobases, which polymer can hybridize by Watson-Crick base pairing to a DNA having the complementary sequence.
  • Nucleobases comprise a base, which is a purine, pyrimidine, or a derivative or analog thereof.
  • Nucleobases include peptide nucleobases, the subunits of peptide nucleic acids, and morpholine nucleobases as well as nucleosides and nucleotides.
  • Nucleosides are nucleobases that contain a pentosefuranosyl moiety, e.g., an optionally substituted riboside or 2′-deoxyriboside.
  • Nucleosides can be linked by one of several linkage moieties, which may or may not contain a phosphorus. Nucleosides that are linked by unsubstituted phosphodiester linkages are termed nucleotides.
  • An oligonucleobase chain has a single 5′ and 3′ terminus, which are the ultimate nucleobases of the polymer.
  • a particular oligonucleobase chain can contain nucleobases of all types.
  • An oligonucleobase compound is a compound comprising one or more oligonucleobase chains that are complementary and hybridized by Watson-Crick base pairing.
  • Nucleobases are either deoxyribo-type or ribo-type.
  • Ribo-type nucleobases are pentosefuranosyl containing nucleobases wherein the 2′ carbon is a methylene substituted with a hydroxyl, alkyloxy or halogen.
  • Deoxyribo-type nucleobases are nucleobases other than ribo-type nucleobases and include all nucleobases that do not contain a pentosefuranosyl moiety.
  • An oligonucleobase strand generically includes both oligonucleobase chains and segments or regions of oligonucleobase chains.
  • An oligonucleobase strand has a 3′ end and a 5′ end. When a oligonucleobase strand is coextensive with a chain, the 3′ and 5′ ends of the strand are also 3′ and 5′ termini of the chain.
  • substantially normal growth of a plant, plant organ, plant tissue or plant cell is defined as a growth rate or rate of cell division of the plant, plant organ, plant tissue, or plant cell that is at least 35%, at least 50%, at least 60%, or at least 75% of the growth rate or rate of cell division in a corresponding plant, plant organ, plant tissue or plant cell expressing the wild type EPSPS protein.
  • substantially normal development of a plant, plant organ, plant tissue or plant cell is defined as the occurrence of one or more developmental events in the plant, plant organ, plant tissue or plant cell that are substantially the same as those occurring in a corresponding plant, plant organ, plant tissue or plant cell expressing the wild type EPSPS protein.
  • plant organs include, but are not limited to, leaves, stems, roots, vegetative buds, floral buds, meristems, embryos, cotyledons, endosperm, sepals, petals, pistils, carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules, ovaries and fruits, or sections, slices or discs taken therefrom.
  • Plant tissues include, but are not limited to, callus tissues, ground tissues, vascular tissues, storage tissues, meristematic tissues, leaf tissues, shoot tissues, root tissues, gall tissues, plant tumor tissues, and reproductive tissues.
  • Plant cells include, but are not limited to, isolated cells with cell walls, variously sized aggregates thereof, and protoplasts.
  • Plants are substantially “tolerant” to glyphosate when they are subjected to it and provide a dose/response curve which is shifted to the right when compared with that provided by similarly subjected non-tolerant like plant.
  • Such dose/response curves have “dose” plotted on the X-axis and “percentage kill”, “herbicidal effect”, etc., plotted on the y-axis. Tolerant plants will require more herbicide than non-tolerant like plants in order to produce a given herbicidal effect.
  • Plants which are substantially “resistant” to the glyphosate exhibit few, if any, necrotic, lytic, chlorotic or other lesions, when subjected to glyphosate at concentrations and rates which are typically employed by the agrochemical community to kill weeds in the field. Plants which are resistant to a herbicide are also tolerant of the herbicide.
  • resistant and tolerant are to be construed as “tolerant and/or resistant” within the context of the present application.
  • EPSPS homolog or any variation therefore refers to an EPSPS gene or EPSPS gene product found in another plant species that performs the same or substantially the same biological function as the EPSPS genes disclosed herein and where the nucleic acid sequences or polypeptide sequences (of the EPSPS gene product) are said to be “identical” or at least 50% similar (also referred to as ‘percent identity’ or ‘substantially identical’) as described below.
  • Two polynucleotides or polypeptides are identical if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.
  • a conservative substitution is given a score between zero and 1.
  • the scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4: 11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
  • phrases “substantially identical,” and “percent identity” in the context of two nucleic acids or polypeptides, refer to sequences or subsequences that have at least 50%, advantageously 60%, preferably 70%, more preferably 80%, and most preferably 90-95% nucleotide or amino acid residue identity when aligned for maximum correspondence over a comparison window as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.
  • two polypeptides can also be “substantially identical” if the two polypeptides are immunologically similar. Thus, overall protein structure may be similar while the primary structure of the two polypeptides display significant variation. Therefore a method to measure whether two polypeptides are substantially identical involves measuring the binding of monoclonal or polyclonal antibodies to each polypeptide. Two polypeptides are substantially identical if the antibodies specific for a first polypeptide bind to a second polypeptide with an affinity of at least one third of the affinity for the first polypeptide. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, 0.4dv. Appl. Math. 2:482 (I 98 I), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'I. Acad. Sci. USA 5 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), by software for alignments such as VECTOR NTI Version #6 by InforMax, Inc.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always>0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff& Henikoff, Proc.
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • a non-transgenic plant or plant cell having one or more mutations in the EPSPS gene is made.
  • the resulting plant has increased resistance or tolerance to a member of the phosphonomethylglycine family such as glyphosate and exhibits substantially normal growth or development of the plant, its organs, tissues or cells, as compared to the corresponding wild-type plant or cell.
  • the mutated gene produces a gene product having a substitution at one or more of the amino acid positions 160, 179, 183, 194, 244, 273 and 454 of the Arabidopsis EPSPS gene AF 360244 product or at an analogous amino acid position in an EPSPS homolog.
  • the mutated plant is resistant to glyphosate and has substantially the same catalytic activity as compared to the wild-type EPSPS protein.
  • in vitro screening can be done in a bacterial system to save time and resources. Growth curves of bacterial colonies expressing candidate mutant EPSPS genes can be generated to evaluate the mutant EPSPS genes in providing a glyphosate resistant phenotype.
  • U.S. Pat. No. 6,870,075 discloses a Salmonella glyphosate resistance assay employing Arabidopsis mutant EPSPS genes transformed into a LacZ-Salmonella typhi strain.
  • the E. coli EPSPS gene also called the AroA gene, can be used to evaluate EPSPS mutants for glyphosate resistance.
  • Preferred amino acid substitutions in the E. coli EPSPS gene (AroA) product include the following:
  • Corresponding amino acid positions in plant species are changed according to the present invention to produce a non-transgenic herbicide resistant plant.
  • Below is a list of some preferred crops which list the amino acid positions in the EPSPS gene to be changed. Preferred amino acid substitutions are listed to the right of the amino acid position number.
  • E. coli is not a plant however it is contemplated in the present invention because the E. coli gene can be mutated in a bacterial cell culture system and then the mutated E. coli gene product (enzyme) can be assayed for enzymatic activity (K l and K m ) that will indicate resistance to glyphosate and function as a necessary enzyme product which is essential in plants. Once a mutated E. coli mutant is identified then that mutation is made in a plant cell employing the recombinagenic oligonucleobases described herein to produce a non-transgenic herbicide resistant plant. For these reasons mutated E. coli and mutated Area proteins are considered part of the present invention.
  • some species have more than one EPSPS gene.
  • one or more of the genes are mutated according to the present invention to make a glyphosate resistant mutant. If the expression levels of the various EPSPS genes is known and is different then it is preferred to mutate the higher expressing EPSPS genes. In a preferred embodiment all of the EPSPS genes in a crop are mutated to make a glyphosate phenotype. For example, canola is known to have four EPSPS genes. Two genes are shown in FIGS. 4 and 5 . A comparison will show a light difference between the two genes.
  • the plant mutated according to the present invention can be of any species of dicotyledonous, monocotyledonous or gymnospermous plant, including any woody plant species that grows as a tree or shrub, any herbaceous species, or any species that produces edible fruits, seeds or vegetables, or any species that produces colorful or aromatic flowers.
  • the plant may be selected from a species of plant from the group consisting of canola, sunflower, tobacco, sugar beet, sweet potato, yam, cotton, maize, wheat, barley, rice, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, onion, soya spp, sugar cane, pea, peanut, field beans, poplar, grape, citrus, alfalfa, rye, oats, turf, and forage grasses, flax, oilseed rape, cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris, lily, and nut producing plants insofar as they are not already specifically mentioned.
  • the recombinagenic oligonucleobase can be introduced into a plant cell using any method commonly used in the art, including but not limited to, microcarriers (biolistic delivery), microfibers (whiskers), electroporation, direct DNA uptake and microinjection.
  • Kmiec I teaches a method for introducing specific genetic alterations into a target gene.
  • the recombinagenic oligonucleobases in Kmiec I and/or Kmiec II contain two complementary strands, one of which contains at least one segment of RNA-type nucleotides (an “RNA segment”) that are base paired to DNA-type nucleotides of the other strand.
  • Kmiec II discloses that purine and pyrimidine base-containing non-nucleotides can be substituted for nucleotides.
  • recombinagenic oligonucleobase is used herein to denote the molecules that can be used in the methods of the present invention and include mixed duplex oligonucleotides, non-nucleotide containing molecules taught in Kmiec II; single stranded oligodeoxynucleotides and other recombinagenic molecules taught in the above noted patents and patent publications.
  • the recombinagenic oligonucleobase is a mixed duplex oligonucleotide in which the RNA-type nucleotides of the mixed duplex oligonucleotide are made RNase resistant by replacing the 2′-hydroxyl with a fluoro, chloro or bromo functionality or by placing a substituent on the 2′-O Suitable substituents include the substituents taught by the Kmiec II. Alternative substituents include the substituents taught by U.S. Pat. No. 5,334,711 (Sproat) and the substituents taught by patent publications EP 629 387 and EP 679 657 (collectively, the Martin Applications), which are incorporated herein by reference.
  • RNA-type nucleotide means a 2′-hydroxyl or 2′-Substituted Nucleotide that is linked to other nucleotides of a mixed duplex oligonucleotide by an unsubstituted phosphodiester linkage or any of the non-natural linkages taught by Kmiec I or Kmiec II.
  • deoxyribo-type nucleotide means a nucleotide having a 2′-H, which can be linked to other nucleotides of a MDON by an unsubstituted phosphodiester linkage or any of the non-natural linkages taught by Kmiec I or Kmiec II.
  • the recombinagenic oligonucleobase is a mixed duplex oligonucleotide that is linked solely by unsubstituted phosphodiester bonds.
  • the linkage is by substituted phosphodiesters, phosphodiester derivatives and non-phosphorus-based linkages as taught by Kmiec II.
  • each RNA-type nucleotide in the mixed duplex oligonucleotide is a 2′-Substituted Nucleotide.
  • 2′-Substituted Ribonucleotides are 2′-fluoro, 2′-methoxy, 2′-propyloxy, 2′-allyloxy, 2′-hydroxylethyloxy, 2′-methoxyethyloxy, 2′-fluoropropyloxy and 2′-trifluoropropyloxy substituted ribonucleotides. More preferred embodiments of 2′-Substituted Ribonucleotides are 2′-fluoro, 2′-methoxy, 2′-methoxyethyloxy, and 2′-allyloxy substituted nucleotides.
  • the mixed duplex oligonucleotide is linked by unsubstituted phosphodiester bonds.
  • RNA segment may not be affected by an interruption caused by the introduction of a deoxynucleotide between two RNA-type trinucleotides, accordingly, the term RNA segment encompasses such an “interrupted RNA segment.”
  • An uninterrupted RNA segment is termed a contiguous RNA segment.
  • an RNA segment can contain alternating RNase-resistant and unsubstituted 2′-OH nucleotides.
  • the mixed duplex oligonucleotides preferably have fewer than 100 nucleotides and more preferably fewer than 85 nucleotides, but more than 50 nucleotides.
  • the first and second strands are Watson-Crick base paired.
  • the strands of the mixed duplex oligonucleotide are covalently bonded by a linker, such as a single stranded hexa, penta or tetranucleotide so that the first and second strands are segments of a single oligonucleotide chain having a single 3′ and a single 5′ end.
  • the 3′ and 5′ ends can be protected by the addition of a “hairpin cap” whereby the 3′ and 5′ terminal nucleotides are Watson-Crick paired to adjacent nucleotides.
  • a second hairpin cap can, additionally, be placed at the junction between the first and second strands distant from the 3′ and 5′ ends, so that the Watson-Crick pairing between the first and second strands is stabilized.
  • the first and second strands contain two regions that are homologous with two fragments of the target EPSPS gene, i.e., have the same sequence as the target gene.
  • a homologous region contains the nucleotides of an RNA segment and may contain one or more DNA-type nucleotides of connecting DNA segment and may also contain DNA-type nucleotides that are not within the intervening DNA segment.
  • the two regions of homology are separated by, and each is adjacent to, a region having a sequence that differs from the sequence of the target gene, termed a “heterologous region.”
  • the heterologous region can contain one, two or three mismatched nucleotides.
  • the mismatched nucleotides can be contiguous or alternatively can be separated by one or two nucleotides that are homologous with the target gene.
  • the heterologous region can also contain an insertion or one, two, three or of five or fewer nucleotides.
  • the sequence of the mixed duplex oligonucleotide may differ from the sequence of the target gene only by the deletion of one, two, three, or five or fewer nucleotides from the mixed duplex oligonucleotide.
  • the length and position of the heterologous region is, in this case, deemed to be the length of the deletion, even though no nucleotides of the mixed duplex oligonucleotide are within the heterologous region.
  • the distance between the fragments of the target gene that are complementary to the two homologous regions is identically the length of the heterologous region when a substitution or substitutions is intended.
  • the heterologous region contains an insertion, the homologous regions are thereby separated in the mixed duplex oligonucleotide farther than their complementary homologous fragments are in the gene, and the converse is applicable when the heterologous region encodes a deletion.
  • RNA segments of the mixed duplex oligonucleotides are each a part of a homologous region, i.e., a region that is identical in sequence to a fragment of the target gene, which segments together preferably contain at least 13 RNA-type nucleotides and preferably from 16 to 25 RNA-type nucleotides or yet more preferably 18-22 RNA-type nucleotides or most preferably 20 nucleotides.
  • RNA segments of the homology regions are separated by and adjacent to, i.e., “connected by” an intervening DNA segment.
  • each nucleotide of the heterologous region is a nucleotide of the intervening DNA segment.
  • An intervening DNA segment that contains the heterologous region of a mixed duplex oligonucleotide is termed a “mutator segment.”
  • the change to be introduced into the target EPSPS gene is encoded by the heterologous region.
  • the change to be introduced into the EPSPS gene may be a change in one or more bases of the EPSPS gene sequence that changes the native amino acid in that position to the desired amino acid.
  • the recombinagenic oligonucleobase is a single stranded oligodeoxynucleotide mutational vector or SSOMV, which is disclosed in International Patent Application PCT/US00/23457, which is incorporated herein by reference in its entirety.
  • SSOMV single stranded oligodeoxynucleotide mutational vector
  • the sequence of the SSOMV is based on the same principles as the mutational vectors described in U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in International Publication Nos.
  • the sequence of the SSOMV contains two regions that are homologous with the target sequence separated by a region that contains the desired genetic alteration termed the mutator region.
  • the mutator region can have a sequence that is the same length as the sequence that separates the homologous regions in the target sequence, but having a different sequence. Such a mutator region will cause a substitution.
  • the nucleotides of the SSOMV are deoxyribonucleotides that are linked by unmodified phosphodiester bonds except that the 3′ terminal and/or 5′ terminal internucleotide linkage or alternatively the two 3′ terminal and/or 5′ terminal internucleotide linkages can be a phosphorothioate or phosphoamidate.
  • an internucleotide linkage is the linkage between nucleotides of the SSOMV and does not include the linkage between the 3′ end nucleotide or 5′ end nucleotide and a blocking substituent, see supra.
  • the length of the SSOMV is between 21 and 55 deoxynucleotides and the lengths of the homology regions are, accordingly, a total length of at least 20 deoxynucleotides and at least two homology regions should each have lengths of at least 8 deoxynucleotides.
  • the SSOMV can be designed to be complementary to either the coding or the non-coding strand of the target gene.
  • both the mutator nucleotides be a pyrimidine.
  • both the mutator nucleotide and the targeted nucleotide in the complementary strand be pyrimidines.
  • Particularly preferred are SSOMV that encode transversion mutations, i.e., a C or T mutator nucleotide is mismatched, respectively, with a C or T nucleotide in the complementary strand.
  • the SSOMV can contain a 5′ blocking substituent that is attached to the 5′ terminal carbons through a linker.
  • the chemistry of the linker is not critical other than its length, which should preferably be at least 6 atoms long and that the linker should be flexible.
  • a variety of non-toxic substituents such as biotin, cholesterol or other steroids or a non-intercalating cationic fluorescent dye can be used.
  • reagents to make SSOMV are the reagents sold as Cy3TM and Cy5TM by Glen Research, Sterling Va., which are blocked phosphoroamidites that upon incorporation into an oligonucleotide yield 3,3,3′,3′-tetramethyl N,N′-isopropyl substituted indomonocarbocyanine and indodicarbocyanine dyes, respectively. Cy3 is the most preferred. When the indocarbocyanine is N-oxyalkyl substituted it can be conveniently linked to the 5′ terminal of the oligodeoxynucleotide through as a phosphodiester with a 5′ terminal phosphate.
  • the chemistry of the dye linker between the dye and the oligodeoxynucleotide is not critical and is chosen for synthetic convenience.
  • the resulting 5′ modification consists of a blocking substituent and linker together which are a N-hydroxypropyl, N′-phosphatidylpropyl 3,3,3′,3′-tetramethyl indomonocarbocyanine.
  • the indocarbocyanine dye is tetra substituted at the 3 and 3′ positions of the indole rings. Without limitation as to theory these substitutions prevent the dye from being an intercalating dye.
  • the identity of the substituents at these positions are not critical.
  • the SSOMV can in addition have a 3′ blocking substituent. Again the chemistry of the 3′ blocking substituent is not critical.
  • the recombinageneic oligonucleotide is a single-stranded oligodeoxynucleotide having a 3′ end nucleotide, a 5′ end nucleotide, having at least 25 deoxynucleotides and not more than 65 deoxynucleotides, and having a sequence comprising at least two regions each of at least 8 deoxynucleotides that are each, respectively, identical to at least two regions of the targeted chromosomal gene, which regions together are at least 24 nucleotides in length, and which regions are separated by at least one nucleotide in the sequence of the targeted chromosomal gene or in the sequence of the oligodeoxynucleotide or both such that the sequence of the oligodeoxynucleotide is not identical to the sequence of the targeted chromosomal gene. See U.S. Pat. No. 6,271,360 which is incorporated herein by reference.
  • microcarriers microspheres
  • U.S. Pat. Nos. 4,945,050; 5,100,792 and 5,204,253 describe general techniques for selecting microcarriers and devices for projecting them.
  • U.S. Pat. Nos. 5,484,956 and 5,489,520 describe the preparation of fertile transgenic corn using microprojectile bombardment of corn callus tissue. The biolistic techniques are also used in transforming immature corn embryos.
  • microcarriers in the methods of the present invention are described in International Publication WO 99/07865.
  • ice cold microcarriers 60 mg/ml
  • mixed duplex oligonucleotide 60 mg/ml
  • 2.5 M CaCl.sub.2 2.5 M spermidine
  • spermidine 0.1 M spermidine
  • the mixture is gently agitated, e.g., by vortexing, for 10 minutes and let stand at room temperature for 10 minutes, whereupon the microcarriers are diluted in 5 volumes of ethanol, centrifuged and resuspended in 100% ethanol.
  • Recombinagenic oligonucleobases can also be introduced into plant cells for the practice of the present invention using microfibers to penetrate the cell wall and cell membrane.
  • U.S. Pat. No. 5,302,523 to Coffee et al. describes the use of 30.times.0.5 ⁇ m and 10.times.0.3 ⁇ m silicon carbide fibers to facilitate transformation of suspension maize cultures of Black Mexican Sweet. Any mechanical technique that can be used to introduce DNA for transformation of a plant cell using microfibers can be used to deliver recombinagenic oligonucleobases for use in making the present EPSPS mutants. The process disclosed by Coffee et al in U.S. Pat. No.
  • 5,302,523 can be employed with regenerable plant cell materials to introduce the present recombinagenic oligonucleobases to effect the mutation of the EPSPS gene whereby a whole mutated plant can be recovered that exhibits the glyphosate resistant phenotype.
  • An illustrative technique for microfiber delivery of a recombinagenic oligonucleobase is as follows: Sterile microfibers (2 ⁇ g) are suspended in 150 ⁇ l of plant culture medium containing about 10.mu.g of a mixed duplex oligonucleotide. A suspension culture is allowed to settle and equal volumes of packed cells and the sterile fiber/nucleotide suspension are vortexed for 10 minutes and plated. Selective media are applied immediately or with a delay of up to about 120 hours as is appropriate for the particular trait.
  • the recombinagenic oligonucleobases can be delivered to the plant cell by electroporation of a protoplast derived from a plant part according to techniques that are well-known to one of ordinary skill in the art. See, e.g., Gallois et al., 1996, in Methods in Molecular Biology 55:89-107, Humana Press, Totowa, N.J.; Kipp et al., 1999, in Methods in Molecular Biology 133:213-221, Humana Press, Totowa, N.J.
  • Recombinagenic oligonucleobases can also be introduced into microspores by electroporation. Upon release of the tetrad, the microspore is uninucleate and thin-walled. It begins to enlarge and develops a germpore before the exine fomms. A microspore at this stage is potentially more amenable to transformation with exogenous DNA than other plant cells.
  • microspore development can be altered in vitro to produce either haploid embryos or embryogenic callus that can be regenerated into plants (Coumans et al., Plant Cell Rep. 7:618-621, 1989; Datta et al., Plant Sci.
  • transformed microspores can be regenerated directly into haploid plants or dihaploid fertile plants upon chromosome doubling by standard methods. See also co-pending application U.S. Ser. No. 09/680,858 entitled Compositions and Methods for Plant Genetic Modification which is incorporated herein by reference.
  • Microspore electroporation can be practiced with any plant species for which microspore culture is possible, including but not limited to plants in the families Graminae, Leguminoceae, Cruciferaceae, Solanaceac, Cucurbitaceae, Rosaccae, Poaceae, Lilaceae, Rutaceae, Vitaceae, including such species as corn ( Zea mays ), wheat ( Triticum aestivum ), rice ( Oryza sativa ), oats, barley, canola ( Brassica napus, Brassica rapa, Brassica oleracea , and Brassica juncea ), cotton ( Gossypium hirsuitum L.), various legume species (e.g., soybean [ Glycine max ], pea [ Pisum sativum ], etc.), grapes [ Vitis vinifera ], and a host of other important crop plants.
  • Microspore embryogenesis both from anther and microspore culture, has been described in more than 170 species, belonging to 68 genera and 28 families of dicotyledons and monocotyledons (Raghavan, Embryogenesis in Agniosperms: A Developmental and Experimental Study, Cambridge University Press, Cambridge, England, 1986; Rhagavan, Cell Differentiation 21:213-226, 1987; Raemakers et al., Euphytica 81:93-107, 1995).
  • Chromosome doubling from microspore or anther culture is a well-established technique for production of double-haploid homozogous plant lines in several crops (Heberle-Bors et al., In vitro pollen cultures: Progress and perspectives. In: Pollen Biotechnology. Gene expression and allergen characterization, vol. 85-109, ed. Mohapatra, S. S., and Knox, R. B., Chapman and Hall, New York, 1996).
  • Microspore electroporation methods are described in Jardinaud et al., Plant Sci. 93:177-184, 1993, and Fennell and Hauptman, Plant Cell Reports 11:567-570, 1992. Methods for electroporation of MDON into plant protoplasts can also be adapted for use in microspore electroporation.
  • the recombinagenic oligonucleobase can be delivered to the plant cell by whiskers or microinjection of the plant cell.
  • the so called whiskers technique is performed essentially as described in Frame et al., 1994, Plant J. 6:941-948.
  • the recombinagenic oligonucleobase is added to the whiskers and used to transform the plant cells.
  • the recombinagenic oligonucleobase may be co-incubated with plasmids comprising sequences encoding proteins capable of forming recombinase complexes in plant cells such that recombination is catalyzed between the oligonucleotide and the target sequence in the EPSPS gene.
  • Plants or plant cells can be tested for resistance or tolerance to a phosphonomethylglycine herbicide using commonly known methods in the art, e.g., by growing the plant or plant cell in the presence of a phosphonomethylglycine herbicide and measuring the rate of growth as compared to the growth rate of control plants in the absence of the herbicide.
  • glyphosate concentrations of from about 0.01 to about 20 mM are employed in selection medium.
  • the underlined nucelobases represent the heterologous region (codon) where the mutation occurs in the canola genome, ie, A.
  • the genoplast is made according to well known techniques and the genoplast is preferably delivered into a canola plant cell via microparticle bombardment, ie, biolistics. Canola plants regenerated that contain the P178A mutant are resistant to glyphosate when applied at commercial rates.
  • SEQ ID 2 VGGAACGCTGGAAC TGC AATGCGAGCATTGACAGCAGCCGTGACTGCH
  • the underlined nucleobases represent the heterologous region (codon) where the mutation occurs in the rice genome, ie, A.
  • the genoplast is made according to well known techniques and the genoplast is preferably delivered into a rice plant cell via microparticle bombardment, ie, biolistics. Rice plants regenerated that contain the P 173A mutant are resistant to glyphosate when applied at commercial rates.
  • T 97 P 101 ⁇ T178A; P182T (T -> A) ACA ⁇ GCA; A 97 ; T 101 (P -> T) CCA ⁇ ACA g. T178A; P182T CCTCGGTAATGCAGGA ACA GCAATGCGT CCA CTTAC CCTCGGTAATGCAGGA gCA GCAATGCGT aCA CTTAC

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)
US12/160,725 2006-01-12 2007-01-10 EPSPS mutants Active 2028-09-20 US8268622B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US12/160,725 US8268622B2 (en) 2006-01-12 2007-01-10 EPSPS mutants

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US75843906P 2006-01-12 2006-01-12
US12/160,725 US8268622B2 (en) 2006-01-12 2007-01-10 EPSPS mutants
PCT/US2007/000591 WO2007084294A2 (en) 2006-01-12 2007-01-10 Epsps mutants

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/000591 A-371-Of-International WO2007084294A2 (en) 2006-01-12 2007-01-10 Epsps mutants

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US13/621,662 Division US20130019349A1 (en) 2006-01-12 2012-09-17 Epsps mutants

Publications (2)

Publication Number Publication Date
US20090307802A1 US20090307802A1 (en) 2009-12-10
US8268622B2 true US8268622B2 (en) 2012-09-18

Family

ID=38288103

Family Applications (3)

Application Number Title Priority Date Filing Date
US12/160,725 Active 2028-09-20 US8268622B2 (en) 2006-01-12 2007-01-10 EPSPS mutants
US13/621,662 Abandoned US20130019349A1 (en) 2006-01-12 2012-09-17 Epsps mutants
US16/040,278 Active US10612035B2 (en) 2006-01-12 2018-07-19 EPSPS mutants

Family Applications After (2)

Application Number Title Priority Date Filing Date
US13/621,662 Abandoned US20130019349A1 (en) 2006-01-12 2012-09-17 Epsps mutants
US16/040,278 Active US10612035B2 (en) 2006-01-12 2018-07-19 EPSPS mutants

Country Status (14)

Country Link
US (3) US8268622B2 (da)
EP (4) EP2465341B1 (da)
JP (1) JP2009523418A (da)
AU (1) AU2007207813B2 (da)
CA (3) CA2636771C (da)
DK (2) DK2465341T3 (da)
ES (2) ES2532112T3 (da)
NZ (4) NZ597682A (da)
PL (2) PL2465341T3 (da)
PT (2) PT2465340E (da)
RU (1) RU2441366C2 (da)
SI (2) SI2465340T1 (da)
UA (1) UA99594C2 (da)
WO (1) WO2007084294A2 (da)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130295638A1 (en) * 2012-02-01 2013-11-07 Dow Agrosciences Llc Synthetic brassica-derived chloroplast transit peptides
WO2015139008A1 (en) 2014-03-14 2015-09-17 Cibus Us Llc Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair
US9303270B2 (en) 2011-07-22 2016-04-05 Ricetec Aktiengesellschaft Rice resistant to HPPD and accase inhibiting herbicides
US9370149B2 (en) 2011-07-22 2016-06-21 Ricetec Aktiengesellschaft Methods and compositions to produce rice resistant to accase inhibitors
US9957515B2 (en) 2013-03-15 2018-05-01 Cibus Us Llc Methods and compositions for targeted gene modification
US20180327774A1 (en) * 2006-01-12 2018-11-15 Cibus Us Llc Epsps mutants
US11130959B2 (en) 2016-08-05 2021-09-28 Ricetec, Inc. Methods and compositions for combinations of mutations associated with herbicide resistance/tolerance in rice
US11542515B2 (en) 2016-02-09 2023-01-03 Cibus Us Llc Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair

Families Citing this family (69)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102791865B (zh) 2009-12-21 2015-07-01 凯津公司 用于转染原生质体的改进技术
AU2013205557B2 (en) * 2012-04-17 2016-04-21 Corteva Agriscience Llc Synthetic brassica-derived chloroplast transit peptides
US12331303B2 (en) 2013-03-15 2025-06-17 Cibus Us Llc Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair
EP4136963B1 (en) * 2013-03-15 2026-01-21 Cibus US LLC Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair
CN120574876A (zh) 2013-08-22 2025-09-02 纳幕尔杜邦公司 使用向导rna/cas内切核酸酶系统的植物基因组修饰及其使用方法
US10329578B2 (en) 2013-10-18 2019-06-25 Pioneer Hi-Bred International, Inc. Glyphosate-N-acetyltransferase (GLYAT) sequences and methods of use
WO2015164457A1 (en) 2014-04-22 2015-10-29 E. I. Du Pont De Nemours And Company Plastidic carbonic anhydrase genes for oil augmentation in seeds with increased dgat expression
BR112017017260A2 (pt) 2015-03-27 2018-04-17 Du Pont construções de dna, vetor, célula, plantas, semente, método de expressão de rna e método de modificação de local alvo
AR105418A1 (es) 2015-05-11 2017-10-04 Two Blades Found Polinucleótidos y métodos para transferir resistencia a la roya asiática del poroto de soja
AU2016263026A1 (en) 2015-05-15 2017-11-09 Pioneer Hi-Bred International, Inc. Guide RNA/Cas endonuclease systems
EP3310803A1 (en) 2015-06-16 2018-04-25 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
CA2990679A1 (en) * 2015-07-02 2017-01-05 Arcadia Biosciences, Inc. Wheat having resistance to glyphosate due to alterations in 5-enol-pyruvylshikimate-3 phosphate synthase
WO2021257206A1 (en) 2020-06-17 2021-12-23 Pioneer Hi-Bred International, Inc. Generating maize plants with enhanced resistance to northern leaf blight
BR112018008134A2 (pt) 2015-10-20 2018-11-06 Pioneer Hi Bred Int método para restaurar a função de um produto gênico não funcional no genoma de uma célula, método para editar uma sequência nucleotídica no genoma de uma célula, planta ou planta de progênie, método para editar uma sequência nucleotídica no genoma de uma célula sem o uso de um molde polinucleotídico de modificação e método para entregar um complexo de rna guia/endonuclease cas numa célula
BR112018008705B1 (pt) 2015-10-30 2023-12-26 Pioneer Hi-Bred International, Inc Método para produzir uma planta transgênica
EP3371313B1 (en) 2015-11-06 2020-10-14 Pioneer Hi-Bred International, Inc. Methods and compositions of improved plant transformation
DK3387134T3 (da) 2015-12-11 2020-12-21 Danisco Us Inc Fremgangsmåder og sammensætninger til øget nukleasemedieret genommodifikation og reducerede virkninger uden for målstedet
EP3390631B1 (en) 2015-12-18 2020-04-08 Danisco US Inc. Methods and compositions for t-rna based guide rna expression
WO2017106414A1 (en) 2015-12-18 2017-06-22 Danisco Us Inc. Methods and compositions for polymerase ii (pol-ii) based guide rna expression
CA3004056C (en) 2015-12-22 2024-01-23 Pioneer Hi-Bred International, Inc. Tissue-preferred promoters and methods of use
WO2017132239A1 (en) 2016-01-26 2017-08-03 Pioneer Hi-Bred International, Inc. Waxy corn
EP3426780A1 (en) 2016-03-11 2019-01-16 Pioneer Hi-Bred International, Inc. Novel cas9 systems and methods of use
WO2017155715A1 (en) 2016-03-11 2017-09-14 Pioneer Hi-Bred International, Inc. Novel cas9 systems and methods of use
EP3426778A1 (en) 2016-03-11 2019-01-16 Pioneer Hi-Bred International, Inc. Novel cas9 systems and methods of use
CA3018384A1 (en) 2016-05-04 2017-11-09 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
CA3022858A1 (en) 2016-06-16 2017-12-21 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
UA127388C2 (uk) 2016-06-24 2023-08-09 Піонір Хай-Бред Інтернешнл, Інк. Регуляторний елемент рослини і спосіб його застосування
EP3478052B1 (en) 2016-07-01 2021-08-25 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
WO2018013333A1 (en) 2016-07-12 2018-01-18 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
EP3535285B1 (en) 2016-11-01 2022-04-06 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
BR112019012339A2 (pt) 2016-12-14 2019-11-26 Pioneer Hi Bred Int polipeptídeo inseticida recombinante, composição, construto de dna, célula hospedeira, planta transgênica, método para inibir o crescimento ou extermínio de uma praga de inseto ou população de praga, polipeptídeo ipd093 quimérico e proteína de fusão
US11213028B2 (en) 2016-12-22 2022-01-04 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
CN106636025B (zh) * 2016-12-28 2017-11-14 四川天豫兴禾生物科技有限公司 一种水稻epsps突变体及其编码基因和应用
WO2018140214A1 (en) 2017-01-24 2018-08-02 Pioneer Hi-Bred International, Inc. Nematicidal protein from pseudomonas
RU2019140646A (ru) 2017-05-11 2021-06-11 Пайонир Хай-Бред Интернэшнл, Инк. Инсектицидные белки и способы их применения
CN111373046A (zh) 2017-09-25 2020-07-03 先锋国际良种公司 组织偏好性启动子和使用方法
CN111465321B (zh) 2017-10-13 2022-12-06 先锋国际良种公司 用于植物细胞的细胞重编程的系统和方法
EP4122947A1 (en) 2017-12-19 2023-01-25 Pioneer Hi-Bred International, Inc. Insecticidal polypeptides and uses thereof
WO2019133371A1 (en) 2017-12-27 2019-07-04 Pioneer Hi-Bred International, Inc. Transformation of dicot plants
EP3759489A1 (en) 2018-03-02 2021-01-06 Pioneer Hi-Bred International, Inc. Plant health assay
US11332752B2 (en) 2018-03-12 2022-05-17 Pioneer Hi-Bred International, Inc. Use of morphogenic factors for the improvement of gene editing
AU2019234562B2 (en) 2018-03-14 2024-08-01 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
CA3092078A1 (en) 2018-03-14 2019-09-19 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
CN111902547B (zh) 2018-03-23 2024-06-14 先锋国际良种公司 鉴定、选择和产生疾病抗性作物的方法
CN111988988A (zh) 2018-04-18 2020-11-24 先锋国际良种公司 鉴定、选择和产生抗白叶枯病水稻的方法
WO2019226508A1 (en) 2018-05-22 2019-11-28 Pioneer Hi-Bred International, Inc. Plant regulatory elements and methods of use thereof
CN112351679B (zh) 2018-06-06 2023-06-13 华中农业大学 鉴定、选择和产生南方玉米锈病抗性作物的方法
CA3097915A1 (en) 2018-06-28 2020-01-02 Pioneer Hi-Bred International, Inc. Methods for selecting transformed plants
EP3821008A1 (en) 2018-07-12 2021-05-19 Keygene N.V. Type v crispr/nuclease-system for genome editing in plant cells
WO2020046701A1 (en) 2018-08-29 2020-03-05 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
EP3628738A1 (en) * 2018-09-25 2020-04-01 KWS SAAT SE & Co. KGaA Method for controlling weed beets and other weeds
US20210395758A1 (en) 2018-10-31 2021-12-23 Pioneer Hi-Bred International, Inc. Compositions and methods for ochrobactrum-mediated plant transformation
EP3874048A1 (en) 2018-11-01 2021-09-08 Keygene N.V. Dual guide rna for crispr/cas genome editing in plants cells
JP2022524615A (ja) 2019-03-11 2022-05-09 パイオニア ハイ－ブレッド インターナショナル， インコーポレイテッド クローン植物の作製方法
EP3947425A1 (en) 2019-03-27 2022-02-09 Pioneer Hi-Bred International, Inc. Plant explant transformation
EP3947696A1 (en) 2019-03-28 2022-02-09 Pioneer Hi-Bred International, Inc. Modified agrobacterium strains and use thereof for plant transformation
BR112021020770A2 (pt) 2019-04-18 2022-01-04 Pioneer Hi Bred Int Fatores de embriogênese para reprogramação celular de uma célula vegetal
US12371706B2 (en) 2019-08-23 2025-07-29 Pioneer Hi-Bred International, Inc. Methods of identifying, selecting, and producing anthracnose stalk rot resistant crops
WO2021076346A1 (en) 2019-10-18 2021-04-22 Pioneer Hi-Bred International, Inc. Maize event dp-202216-6 and dp-023211-2 stack
CN111153974A (zh) 2020-01-15 2020-05-15 华中农业大学 玉米抗病基因和分子标记及其应用
BR112022020714A2 (pt) 2020-04-15 2022-12-20 Pioneer Hi Bred Int Identificação de gene efetor de patógeno vegetal e de resistência à doença, composições e métodos de uso
BR112022027035A2 (pt) 2020-07-14 2023-04-11 Pioneer Hi Bred Int Proteínas inseticidas e métodos para uso das mesmas
CN116096903A (zh) 2020-08-10 2023-05-09 先锋国际良种公司 植物调节元件及其使用方法
WO2022072335A2 (en) 2020-09-30 2022-04-07 Pioneer Hi-Bred International, Inc. Rapid transformation of monocot leaf explants
CN116635529A (zh) 2020-10-21 2023-08-22 先锋国际良种公司 双单倍体诱导物
CA3197681A1 (en) 2020-10-21 2022-04-28 Pioneer Hi-Bred International, Inc. Parthenogenesis factors and methods of using same
CN115216554A (zh) 2021-04-16 2022-10-21 华中农业大学 植物病原体效应子和疾病抗性基因鉴定、组合物和使用方法
US20250019717A1 (en) 2021-08-06 2025-01-16 Kws Vegetables B.V. Durable downy mildew resistance in spinach
EP4499841A1 (en) 2022-03-25 2025-02-05 Pioneer Hi-Bred International, Inc. Methods of parthenogenic haploid induction and haploid chromosome doubling

Citations (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4545060A (en) 1983-09-19 1985-10-01 Northern Telecom Limited Decision feedback adaptive equalizer acting on zero states following a non-zero state
US4945050A (en) 1984-11-13 1990-07-31 Cornell Research Foundation, Inc. Method for transporting substances into living cells and tissues and apparatus therefor
US5100792A (en) 1984-11-13 1992-03-31 Cornell Research Foundation, Inc. Method for transporting substances into living cells and tissues
US5145783A (en) 1987-05-26 1992-09-08 Monsanto Company Glyphosate-tolerant 5-endolpyruvyl-3-phosphoshikimate synthase
US5204253A (en) 1990-05-29 1993-04-20 E. I. Du Pont De Nemours And Company Method and apparatus for introducing biological substances into living cells
US5302523A (en) 1989-06-21 1994-04-12 Zeneca Limited Transformation of plant cells
US5310667A (en) 1989-07-17 1994-05-10 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases
US5312910A (en) 1987-05-26 1994-05-17 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthase
US5334711A (en) 1991-06-20 1994-08-02 Europaisches Laboratorium Fur Molekularbiologie (Embl) Synthetic catalytic oligonucleotide structures
EP0629387A1 (en) 1993-06-11 1994-12-21 SILCA S.p.A. Shaped sanitary towel for incontinence
US5484956A (en) 1990-01-22 1996-01-16 Dekalb Genetics Corporation Fertile transgenic Zea mays plant comprising heterologous DNA encoding Bacillus thuringiensis endotoxin
US5489520A (en) 1990-04-17 1996-02-06 Dekalb Genetics Corporation Process of producing fertile transgenic zea mays plants and progeny comprising a gene encoding phosphinothricin acetyl transferase
US5565350A (en) 1993-12-09 1996-10-15 Thomas Jefferson University Compounds and methods for site directed mutations in eukaryotic cells
WO1997004103A2 (fr) 1995-07-19 1997-02-06 Rhone-Poulenc Agrochimie 5-enol pyruvylshikimate-3-phosphate synthase mutee, gene codant pour cette proteine et plantes transformees contenant ce gene
US5731181A (en) 1996-06-17 1998-03-24 Thomas Jefferson University Chimeric mutational vectors having non-natural nucleotides
US5750673A (en) 1994-04-27 1998-05-12 Novartis Corporation Nucleosides with 2'-O-modifications
US5760012A (en) 1996-05-01 1998-06-02 Thomas Jefferson University Methods and compounds for curing diseases caused by mutations
US5780296A (en) 1995-01-17 1998-07-14 Thomas Jefferson University Compositions and methods to promote homologous recombination in eukaryotic cells and organisms
US5804425A (en) 1990-08-31 1998-09-08 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
WO1998044140A1 (en) 1997-04-03 1998-10-08 Dekalb Genetics Corporation Glyphosate resistant maize lines
WO1998049350A1 (en) 1997-04-30 1998-11-05 Regents Of The University Of Minnesota In vivo use of recombinagenic oligonucleobases to correct genetic lesions in hepatocytes
WO1998054330A1 (en) 1997-05-28 1998-12-03 Zeneca Limited Methods of in situ modification of plant genes
US5866775A (en) 1990-09-28 1999-02-02 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases
WO1999007865A1 (en) 1997-08-05 1999-02-18 Kimeragen, Inc. The use of mixed duplex oligonucleotides to effect localized genetic changes in plants
US5888983A (en) 1996-05-01 1999-03-30 Thomas Jefferson University Method and oligonucleobase compounds for curing diseases caused by mutations
KR19990029091A (ko) 1995-07-19 1999-04-15 제닌 페트릭 식물의 형질전환용으로 사용될 수 있는 키메라 유전자에서조절영역으로 사용할 수 있는 단리 디엔에이 서열
WO1999040789A1 (en) 1998-02-12 1999-08-19 Regents Of The University Of Minnesota Methods and compounds for the genetic treatment of hyperlipidemia
WO1999058723A1 (en) 1998-05-12 1999-11-18 Kimeragen, Inc. Heteroduplex mutational vectors and use thereof in bacteria
WO1999058702A1 (en) 1998-05-12 1999-11-18 Kimeragen, Inc. Cell-free chimeraplasty and eukaryotic use of heteroduplex mutational vectors
KR20000023830A (ko) 1996-07-16 2000-04-25 제닌 페트릭 다수의 제초제 내성 유전자를 갖는 키메라 유전자, 다수의 제초제 내성 식물세포 및 식물
WO2001000843A2 (en) 1999-06-25 2001-01-04 Basf Aktiengesellschaft Corynebacterium glutamicum genes encoding metabolic pathway proteins
WO2001015740A1 (en) 1999-08-27 2001-03-08 Valigen (Us), Inc. Single-stranded oligodeoxynucleotide mutational vectors
WO2001024615A1 (en) * 1999-10-07 2001-04-12 Valigen (Us), Inc. Non-transgenic herbicide resistant plants
US20030084473A1 (en) 2001-08-09 2003-05-01 Valigen Non-transgenic herbicide resistant plants
KR20050064875A (ko) 2003-12-24 2005-06-29 한국생명공학연구원 한세눌라 폴리모파의 시키메이트 경로 다기능 효소를코딩하는 유전자 및 이를 이용한 코리스메이트의 생합성방법
WO2009002150A1 (en) 2007-06-22 2008-12-31 Keygene N.V. Targeted nucleotide exchange with improved modified oligonucleotides
WO2009082190A1 (en) 2007-12-21 2009-07-02 Keygene N.V. An improved mutagenesis method using polyethylene glycol mediated introduction of mutagenic nucleobases into plant protoplasts
WO2010036771A2 (en) 2008-09-26 2010-04-01 Basf Agrochemical Products B.V. Herbicide-resistant ahas-mutants and methods of use

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4971908A (en) * 1987-05-26 1990-11-20 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthase
JPH0651777B2 (ja) * 1989-02-21 1994-07-06 株式会社日立製作所 熱硬化性樹脂組成物及びそれを用いたコイル、パネル
US5593874A (en) * 1992-03-19 1997-01-14 Monsanto Company Enhanced expression in plants
US5454060A (en) * 1994-08-03 1995-09-26 Mcdermott; Neal J. Foot dryer
RU2235778C2 (ru) * 1999-04-29 2004-09-10 Синджента Лимитед Выделенный полинуклеотид, способный придавать растению устойчивость или толерантность к глифосатному гербициду, вектор, способ получения растений, толерантных или устойчивых к глифосатному гербициду, способ регенерации трансформированного растения и способ селективной борьбы с сорняками
US7045684B1 (en) * 2002-08-19 2006-05-16 Mertec, Llc Glyphosate-resistant plants
CN102094032B (zh) * 2004-04-30 2014-02-26 美国陶氏益农公司 新除草剂抗性基因
CA2636771C (en) * 2006-01-12 2016-05-24 Greg F.W. Gocal Epsps mutants

Patent Citations (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4545060A (en) 1983-09-19 1985-10-01 Northern Telecom Limited Decision feedback adaptive equalizer acting on zero states following a non-zero state
US4945050A (en) 1984-11-13 1990-07-31 Cornell Research Foundation, Inc. Method for transporting substances into living cells and tissues and apparatus therefor
US5100792A (en) 1984-11-13 1992-03-31 Cornell Research Foundation, Inc. Method for transporting substances into living cells and tissues
US5312910A (en) 1987-05-26 1994-05-17 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthase
US5145783A (en) 1987-05-26 1992-09-08 Monsanto Company Glyphosate-tolerant 5-endolpyruvyl-3-phosphoshikimate synthase
US5302523A (en) 1989-06-21 1994-04-12 Zeneca Limited Transformation of plant cells
US5310667A (en) 1989-07-17 1994-05-10 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases
US5484956A (en) 1990-01-22 1996-01-16 Dekalb Genetics Corporation Fertile transgenic Zea mays plant comprising heterologous DNA encoding Bacillus thuringiensis endotoxin
US5489520A (en) 1990-04-17 1996-02-06 Dekalb Genetics Corporation Process of producing fertile transgenic zea mays plants and progeny comprising a gene encoding phosphinothricin acetyl transferase
US5204253A (en) 1990-05-29 1993-04-20 E. I. Du Pont De Nemours And Company Method and apparatus for introducing biological substances into living cells
US5804425A (en) 1990-08-31 1998-09-08 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
US5866775A (en) 1990-09-28 1999-02-02 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases
US5334711A (en) 1991-06-20 1994-08-02 Europaisches Laboratorium Fur Molekularbiologie (Embl) Synthetic catalytic oligonucleotide structures
EP0629387A1 (en) 1993-06-11 1994-12-21 SILCA S.p.A. Shaped sanitary towel for incontinence
US5565350A (en) 1993-12-09 1996-10-15 Thomas Jefferson University Compounds and methods for site directed mutations in eukaryotic cells
US5756325A (en) 1993-12-09 1998-05-26 Thomas Jefferson University Compounds and methods for site directed mutations in eukaryotic cells
US5871984A (en) 1993-12-09 1999-02-16 Thomas Jefferson University Compounds and methods for site directed mutations in eukaryotic cells
US5750673A (en) 1994-04-27 1998-05-12 Novartis Corporation Nucleosides with 2'-O-modifications
EP0679657B1 (de) 1994-04-27 2003-07-09 Novartis AG Nukleoside und Oligonukleotide mit 2'-Ethergruppen
US5780296A (en) 1995-01-17 1998-07-14 Thomas Jefferson University Compositions and methods to promote homologous recombination in eukaryotic cells and organisms
US5945339A (en) 1995-01-17 1999-08-31 Cornell Research Foundation, Inc. Methods to promote homologous recombination in eukaryotic cells and organisms
KR19990029091A (ko) 1995-07-19 1999-04-15 제닌 페트릭 식물의 형질전환용으로 사용될 수 있는 키메라 유전자에서조절영역으로 사용할 수 있는 단리 디엔에이 서열
US6566587B1 (en) 1995-07-19 2003-05-20 Bayer Cropscience S.A. Mutated 5-enolpyruvylshikimate-3-phosphate synthase, gene coding for said protein and transformed plants containing said gene
WO1997004103A2 (fr) 1995-07-19 1997-02-06 Rhone-Poulenc Agrochimie 5-enol pyruvylshikimate-3-phosphate synthase mutee, gene codant pour cette proteine et plantes transformees contenant ce gene
US5760012A (en) 1996-05-01 1998-06-02 Thomas Jefferson University Methods and compounds for curing diseases caused by mutations
US5888983A (en) 1996-05-01 1999-03-30 Thomas Jefferson University Method and oligonucleobase compounds for curing diseases caused by mutations
US5731181A (en) 1996-06-17 1998-03-24 Thomas Jefferson University Chimeric mutational vectors having non-natural nucleotides
US5795972A (en) 1996-06-17 1998-08-18 Thomas Jefferson University Chimeric mutational vectors having non-natural nucleotides
KR20000023830A (ko) 1996-07-16 2000-04-25 제닌 페트릭 다수의 제초제 내성 유전자를 갖는 키메라 유전자, 다수의 제초제 내성 식물세포 및 식물
WO1998044140A1 (en) 1997-04-03 1998-10-08 Dekalb Genetics Corporation Glyphosate resistant maize lines
WO1998049350A1 (en) 1997-04-30 1998-11-05 Regents Of The University Of Minnesota In vivo use of recombinagenic oligonucleobases to correct genetic lesions in hepatocytes
WO1998054330A1 (en) 1997-05-28 1998-12-03 Zeneca Limited Methods of in situ modification of plant genes
GB2326163A (en) * 1997-05-28 1998-12-16 Zeneca Ltd In situ modification of plant genes
WO1999007865A1 (en) 1997-08-05 1999-02-18 Kimeragen, Inc. The use of mixed duplex oligonucleotides to effect localized genetic changes in plants
WO1999040789A1 (en) 1998-02-12 1999-08-19 Regents Of The University Of Minnesota Methods and compounds for the genetic treatment of hyperlipidemia
WO1999058723A1 (en) 1998-05-12 1999-11-18 Kimeragen, Inc. Heteroduplex mutational vectors and use thereof in bacteria
WO1999058702A1 (en) 1998-05-12 1999-11-18 Kimeragen, Inc. Cell-free chimeraplasty and eukaryotic use of heteroduplex mutational vectors
US6004804A (en) 1998-05-12 1999-12-21 Kimeragen, Inc. Non-chimeric mutational vectors
US6010907A (en) 1998-05-12 2000-01-04 Kimeragen, Inc. Eukaryotic use of non-chimeric mutational vectors
WO2001000843A2 (en) 1999-06-25 2001-01-04 Basf Aktiengesellschaft Corynebacterium glutamicum genes encoding metabolic pathway proteins
US6271360B1 (en) 1999-08-27 2001-08-07 Valigen (Us), Inc. Single-stranded oligodeoxynucleotide mutational vectors
WO2001015740A1 (en) 1999-08-27 2001-03-08 Valigen (Us), Inc. Single-stranded oligodeoxynucleotide mutational vectors
WO2001024615A1 (en) * 1999-10-07 2001-04-12 Valigen (Us), Inc. Non-transgenic herbicide resistant plants
US6870075B1 (en) 1999-10-07 2005-03-22 Valigen (Us), Inc. Methods of making non-transgenic herbicide resistant plants
US20050177899A1 (en) 1999-10-07 2005-08-11 Beetham Peter R. Non-transgenic herbicide resistant plants
US20030084473A1 (en) 2001-08-09 2003-05-01 Valigen Non-transgenic herbicide resistant plants
KR20050064875A (ko) 2003-12-24 2005-06-29 한국생명공학연구원 한세눌라 폴리모파의 시키메이트 경로 다기능 효소를코딩하는 유전자 및 이를 이용한 코리스메이트의 생합성방법
WO2009002150A1 (en) 2007-06-22 2008-12-31 Keygene N.V. Targeted nucleotide exchange with improved modified oligonucleotides
WO2009082190A1 (en) 2007-12-21 2009-07-02 Keygene N.V. An improved mutagenesis method using polyethylene glycol mediated introduction of mutagenic nucleobases into plant protoplasts
WO2010036771A2 (en) 2008-09-26 2010-04-01 Basf Agrochemical Products B.V. Herbicide-resistant ahas-mutants and methods of use

Non-Patent Citations (52)

* Cited by examiner, † Cited by third party
Title
Agarwal, et al, Nucleotide replacement at two sites can be directed by modified single-stranded oligonucleotied in vitro and in vivo, Biomolecular Engineering, (2003), 20:7-20 [D17].
Beetham et al., 1999, "A Tool for Functional Plant Genomics; Chimeric RNA/DNA Oligonucleotides Cause in Vivo Gene-Specific Mutations," Proc. Nat'l. Acad. Sci. USA, 96:8774-8778. [D5].
Comai, et al, An altered aroA gene product confers resistance to the herbicide glyphosate, Science, (1983), 221:370-371 [D12].
Communication pursuant to Article 94(3) EPC dated Jul. 22, 2011 for related EPO Patent Application No. 07716464.8.
Database entry for 3-phosphoshikimate 1-carboxyvinyltransferase-Bacillus subtilis from website http://www.uniprot.org/uniprot/P20691-last-updated Feb. 8, 2011. [D18].
Dyer et al., Glyphosate Tolerance in Tobacco (Nicotiana tabacum L.) Plant Physiol., vol. 88, pp. 661-666 (1988). [D27].
Escorial et al., "In vitro Culture Selection Increases Glyphosate Tolerance in Barley," Plant Cell Tissue and Organ Culture, vol. 46, pp. 179-186 (1996). [D28].
European Opposition documents cited in related European Patent No. 1223799, dated Jan. 27, 2012.
Experimental result II Keygene, 3 pgs. [D25].
Experimental Results by Keygene NV-D14-TNE events tomato tobacco ALS P194 508691 (cited in the Notice of Opposition to a European Patent in application EP 00970716, dated Sep. 9, 2010, at pp. 37-38 [D14], 2 pages.
Frame et al., 1994, "Production of Fertile Transgenic Maize Plants by Sillicon Carbide Whisker-Mediated Transformation," Plant J., 6:941-948.
Funke et al., Structural Basis of Glyphosate Resistance Resulting from the Double Mutation Thr97 ->Ile and Pro101 ->Ser in 5-Enolpyruvylshikimate-3-phosphate Synthase from Escherichia coli, Journal of Biological Chemistry (2009) 284:9854-9860 [D19].
Funke et al., Structural Basis of Glyphosate Resistance Resulting from the Double Mutation Thr97 →Ile and Pro101 →Ser in 5-Enolpyruvylshikimate-3-phosphate Synthase from Escherichia coli, Journal of Biological Chemistry (2009) 284:9854-9860 [D19].
Gallois et al, 1996, "Electroporation of Tobacco Leaf Protoplasts Using Plasmid DNA or Total Genomic DNA," Methods in Molecular Biology, 55:89-107, Humana Press, Totowa, NJ.
Goldsbrough et al., "Gene amplification in glyphosate tolerant tobacco cells," Plant Science, vol. 72, pp. 53-62 (1990). [D29].
Hegele, et al, Simultaneous targeted exchange of two nucleotides by single stranded oligonucleotides clusters within a region of about fourteen nucleotides, BMC Mol Biol, 2008, 9:14 [D15].
Henner et al., "The Organization and Nucleotide Sequence of the bacillus subtilis hisH, tyrA and aroE genes," Gene 49, pp. 147-152 (1986). [D18A].
Hohn et al., "Gene Therapy in Plants," Proc. Natl. Acad. Sci., vol. 96, pp. 8321-8323 (1999). [D4].
International Search Report dated Jul. 6, 2007 for related application PCT/US2007/000591.
Kenner, et al, Concurrent targeted exchange of three bases in mammalian hprt by oligonucleotides, Biochem Biophys Res Comm, (2004), 321:1017-1023 [D16].
Kipp et al., 1999, "Gene-Targeting in Plants via Site-Directed Mutagenesis," Methods in Molecular Biology, 133:213-221, Humana Press, Totowa, NJ.
Kishore and Shah, "Amino Acid Biosynthesis Inhibitors as Herbicides," Ann. Rev. Biochem., 57:627-663, 1988.
Kishore et al., 1986, abstract, "Isolation, Purification and Characterization of a Glyphosate Tolerant Mutant E. coil EPSP Syntase," Fed. Proc., 45:1506.
Kochevenko, et al, Chimeric RNA/DNA oligonucleotide-based site-specific modification of the tobacco acetelactate syntase gene, Plant Physiology, (2003), 132:174-184 [Exh. F].
Majumder et al., "Background-minized Cassette Mutagenesis by PCR Using Cassette-specific Selection Markers: A Useful General Approach for Studying Structure-Function Relationships of Multisubstrate Enzymes," PCR Methods and Applications, vol. 4, pp. 212-218 (1995). [D24].
Meredith, "On Being Selective: Mutants from Cultured Cells," Plant Molecular Biol. Reporter, vol. 1:3, pp. 105-110 (1983). [D30].
Ng et al 2003, Weed Science 43(2): 108-115. *
Notice of Opposition to a European Patent in application EP 00970716, dated Sep. 9, 2010, 45 pages (including Exh. 1-Declaration and CV of Peter Beetham dated (2007 at pp. 2-3)).
Oh, et al, Oligonucleotide-directed plant gene targeting, Curr Opin Biotech, (2001), 12:169-172 [Exh. E].
Okuzaki, et al, Chimeric RNA/DNA oligonucleotide-directed gene targeting in rice,Plant Cell Rep, (2004), 22:509-512 [Exh. G].
Padgett et al., "Site-directed Mutagenesis of a Conserved Region of the 5-Encolypyruvylshikimate-3-phosphate Synthase Active Site," The Journ. of Biol. Chem., vol. 266, No. 33, pp. 22364-22369 (1991).
Padgette et al., "Herbicide-Resistant Crops," CRC Lewis publishers, Duke Ed., Chap. 4, pp. 53-84 and 4 pgs. of pictures (1996). [D23].
Rice et al., "Genetic Repair of Mutations in Plant Cell-free Extracts Directed by Specific Chimeric Oligonucleotides1," Jun. 2000, 123:427-437. [Exh. C].
Rice, et al, The potential of nucleic acid repair in functional genomics, Plant Physiology, (2001), 19:321-326 [Exh. D].
Ruiter, et al, Spontaneous mutation frequency in plants obscures the effect of chimeraplasty, Plant Molecular Bio, (2003), 53:715-729 [D7].
Schultz et al., 1984, "Insensitivity of 5-Enolpyruvylshikimic Acid-3-Phosphate Synthase to Glyphosphate Confers Resistance to this Herbicide in a Strain of Aerobacter aerogenes," Arch. Microbiol., 137:121-123.
Selection of protoplasts for herbicide tolerance: different outcomes of the selection, Jan. 19, 2012, 3 pgs. [D26].
Selvapandiyan, et al, Point mutation of a conserved arginine to lysine introduces hypersensitivity to inhibition by glyphosate, FEBS Lett, (1995), 374:253-256 [D8].
Shah et al, 1986, "Engineering Herbicide Tolerance in Transgenic Plants," Science, 233:478-481.
Shuttleworth et al., "Site-Directed Mutagenesis of Putative Active Site Residues of 5-Enolpyruvylshikimate-3-phosphate Synthase," Biochemistry, vol. 38, pp. 296-302 (1999). [D21].
Shyr et al., "Glyphosate selected amplification of the 5-enolypyruvylshikimate-3-phosphate synthase gene in cultured carrot cells," Mol. Gen. Genet., vol. 232, pp. 377-382 (1992). [D31].
Singer et al., "Selection of Glyphosate-Tolerant Tobacco Calli and the Expression of this Tolerance in Renegerated Plants," Plant Physiol., vol. 78, pp. 411-416 (1985). [D32].
Sost and Amrhein, 1990, "Subsititution of Gly-96 to Ala in the 5-Enolpyruvylshikimate 3-Phosphate Synthase of Klebsiella pneumoniae Results in a Greatly Reduced Affinity for the Herbicide Glyphosphate," Arch. Biochem. Biophys., 282:433-436.
Stalker, et al, A single amino acid sustitution in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase confers resitance to the herbicide glyphosate, J Bio Chem, (1985), 260(8):4724-4728 [D9].
Stallings et al., "Structure and topological symmetry of the glyphosate target 5-enol-pyruvylshikimate-3-phosphate synthase: A distinctive protein fold," Proc. Natl. Acad. Sci., vol. 88, pp. 5046-5050 (1991). [D35].
Supplementary European Search Report issued Sep. 14, 2009 for EPO Patent Application No. 07716464.
Townsend et al., "High-frequency modification of plant genes using engineered zinc-finger nucleases," Nature, vol. 459, pp. 442-445 (2009). [D33].
U.S. Appl. No. 09/680,858, filed Oct. 6, 2000, Beetham.
Widholm et al., "Glyphosate selection of gene amplification in suspension cultures of 3 plant species," Physiologia Plantarum, vol. 112, pp. 540-545 (2001). [D34].
Zhang, et al, Plant gene targeting and gene replacement: application to crop genetic improvement, Chin J Agr Biot, (2008), 5(2):93-99 [D10].
Zhu et al., 2000, "Engineering Herbicide-Resistant Maize Using Chimeric RNA/DNA Oligonucleotides," Nat. Biotech, 18:555-558. [Exh. B].
Zhu et al., Jul. 1999, "Targeted Manipulation of Maize Genes in vivo Using Chimeric RNA/DNA Oligonucleotides," Proc. Nat'l. Acad. Sci., Plant Biology, 96:8768-8773. [D6].

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180327774A1 (en) * 2006-01-12 2018-11-15 Cibus Us Llc Epsps mutants
US10612035B2 (en) * 2006-01-12 2020-04-07 Cibus Us Llc EPSPS mutants
US9370149B2 (en) 2011-07-22 2016-06-21 Ricetec Aktiengesellschaft Methods and compositions to produce rice resistant to accase inhibitors
US9994862B2 (en) 2011-07-22 2018-06-12 Ricetec, Inc. Rice resistant to HPPD and ACCase inhibiting herbicides
US9303270B2 (en) 2011-07-22 2016-04-05 Ricetec Aktiengesellschaft Rice resistant to HPPD and accase inhibiting herbicides
US20130295638A1 (en) * 2012-02-01 2013-11-07 Dow Agrosciences Llc Synthetic brassica-derived chloroplast transit peptides
US9493781B2 (en) * 2012-02-01 2016-11-15 Dow Agrosciences Llc Synthetic brassica-derived chloroplast transit peptides
US10428339B2 (en) 2012-02-01 2019-10-01 Dow Agrosciences Llc Synthetic Brassica-derived chloroplast transit peptides
US9957515B2 (en) 2013-03-15 2018-05-01 Cibus Us Llc Methods and compositions for targeted gene modification
US10954522B2 (en) 2013-03-15 2021-03-23 Cibus Us Llc Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair
US11761011B2 (en) 2013-03-15 2023-09-19 Cibus Us Llc Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair
WO2015139008A1 (en) 2014-03-14 2015-09-17 Cibus Us Llc Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair
EP4357452A2 (en) 2014-03-14 2024-04-24 Cibus US LLC Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair
US11542515B2 (en) 2016-02-09 2023-01-03 Cibus Us Llc Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair
US11130959B2 (en) 2016-08-05 2021-09-28 Ricetec, Inc. Methods and compositions for combinations of mutations associated with herbicide resistance/tolerance in rice

Also Published As

Publication number Publication date
WO2007084294A2 (en) 2007-07-26
US10612035B2 (en) 2020-04-07
NZ613267A (en) 2015-02-27
SI2465340T1 (sl) 2015-04-30
WO2007084294A3 (en) 2007-09-13
CA2636771C (en) 2016-05-24
EP2923563B1 (en) 2024-09-18
DK2465341T3 (da) 2015-03-02
RU2441366C2 (ru) 2012-02-10
EP2923563A2 (en) 2015-09-30
EP2465340B1 (en) 2014-12-17
AU2007207813B2 (en) 2013-09-12
JP2009523418A (ja) 2009-06-25
CA3077776C (en) 2022-10-04
NZ597682A (en) 2014-02-28
PT2465340E (pt) 2015-03-11
ES2532112T3 (es) 2015-03-24
US20130019349A1 (en) 2013-01-17
EP1978799A2 (en) 2008-10-15
CA2636771A1 (en) 2007-07-26
RU2008133046A (ru) 2010-02-20
UA99594C2 (en) 2012-09-10
DK2465340T3 (da) 2015-03-02
NZ570009A (en) 2012-02-24
CA2923605C (en) 2020-06-02
EP2465341B1 (en) 2014-12-17
PL2465340T3 (pl) 2015-05-29
PT2465341E (pt) 2015-03-09
EP1978799A4 (en) 2009-11-11
EP2465341A1 (en) 2012-06-20
ES2531882T3 (es) 2015-03-20
NZ704098A (en) 2015-02-27
PL2465341T3 (pl) 2015-06-30
SI2465341T1 (sl) 2015-04-30
US20180327774A1 (en) 2018-11-15
CA2923605A1 (en) 2007-07-26
EP2923563A3 (en) 2015-10-14
AU2007207813A8 (en) 2009-09-24
CA3077776A1 (en) 2007-07-26
WO2007084294A8 (en) 2009-07-02
EP2465340A1 (en) 2012-06-20
US20090307802A1 (en) 2009-12-10
AU2007207813A1 (en) 2007-07-26

Similar Documents

Publication Publication Date Title
US10612035B2 (en) EPSPS mutants
US11160224B2 (en) Non-transgenic herbicide resistant plants
US20030084473A1 (en) Non-transgenic herbicide resistant plants
US20230141977A1 (en) Targeted gene modification using oligonucleotide-mediated gene repair

Legal Events

Date Code Title Description
AS Assignment

Owner name: CIBUS, LLC, CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GOCAL, GREG F.W.;KNUTH, MARK E.;BEETHAM, PETER R.;REEL/FRAME:021806/0868;SIGNING DATES FROM 20080807 TO 20080808

Owner name: CIBUS, LLC, CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GOCAL, GREG F.W.;KNUTH, MARK E.;BEETHAM, PETER R.;SIGNING DATES FROM 20080807 TO 20080808;REEL/FRAME:021806/0868

AS Assignment

Owner name: CIBUS US LLC, CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CIBUS LLC;REEL/FRAME:022760/0145

Effective date: 20090101

AS Assignment

Owner name: CIBUS US LLC, CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CIBUS US LLC;REEL/FRAME:026138/0359

Effective date: 20110412

Owner name: INCIMA US LLC, CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CIBUS US LLC;REEL/FRAME:026138/0359

Effective date: 20110412

FEPP Fee payment procedure

Free format text: PAYOR NUMBER ASSIGNED (ORIGINAL EVENT CODE: ASPN); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY

STCF Information on status: patent grant

Free format text: PATENTED CASE

FPAY Fee payment

Year of fee payment: 4

AS Assignment

Owner name: CIBUS US LLC, CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:INCIMA US LLC;REEL/FRAME:047441/0178

Effective date: 20181029

MAFP Maintenance fee payment

Free format text: PAYMENT OF MAINTENANCE FEE, 8TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1552); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

Year of fee payment: 8

FEPP Fee payment procedure

Free format text: ENTITY STATUS SET TO SMALL (ORIGINAL EVENT CODE: SMAL); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY

MAFP Maintenance fee payment

Free format text: PAYMENT OF MAINTENANCE FEE, 12TH YR, SMALL ENTITY (ORIGINAL EVENT CODE: M2553); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY

Year of fee payment: 12